体外扩增人脐血造血干祖细胞方法初步研究

发布时间：2018-02-21 02:57

本文关键词： 造血干细胞造血祖细胞体外扩增三维培养藻酸盐腺病毒载体 p18ink4c CKI siRNA　出处：《复旦大学》2008年博士论文　论文类型：学位论文

【摘要】：脐血是临床上良好的造血干祖细胞来源之一。然而,单份脐血所能提供的造血干祖细胞数量较少,限制了脐血移植的应用范围。为了满足青少年和成人患者的需要,必须在移植前体外扩增脐血的造血干祖细胞数量。目前常规的体外扩增造血干祖细胞的实验方法是将造血干祖细胞单个悬浮培养,依赖于较高剂量多种细胞因子的联合作用。在这样的培养体系里扩增后的细胞无法长期重建造血。因此,发展新的体外扩增造血干祖细胞的方法是十分必要的。第一部分,藻酸盐三维培养系统体外扩增人脐血造血干祖细胞造血干祖细胞在体内生存于骨髓微环境中,造血细胞之间、造血细胞与基质细胞之间、细胞与细胞外基质之间的相互作用是维持干细胞自我更新能力的因素之一。三维培养体系旨在模拟骨髓微环境而设计。我们的实验设计藻酸盐三维培养体系,包装脐血单个核细胞,培养于培养皿静止系统和Synthecon RWV旋转培养系统中,与常规培养体系(二维系统)相比较。结果显示常规培养体系在低浓度细胞因子培养脐血单个核细胞,细胞总数下降,CD34+细胞明显减少。而三维静止培养体系在低浓度细胞因子培养条件下,细胞总数上升,CD34+细胞明显增加。三维旋转培养体系则可在更低浓度细胞因子作用下得到相类似的结果。在三维培养体系扩增的细胞其粒巨系克隆形成细胞亦明显增多,移植NOD/SCID小鼠可在移植细胞总数少的情况下,在小鼠体内更好植活,重建造血。第二部分,下调细胞周期素依赖激酶抑制因子p18ink4c对脐血造血干祖细胞的作用细胞的增殖分化是受到细胞周期的严格调控的。调控细胞周期的有三大类重要分了,细胞周期素,细胞周期素依赖激酶和细胞周期素依赖激酶抑制因子CKI。p18ink4c属于CKI,它作用于细胞周期G1期早期,抑制cyclinD-CDK4/6复合物的活性,使细胞停留于GO期。p18ink4c-/-小鼠表现为普遍的组织增生和器官肿大,正常小鼠移植p18ink4c-/-小鼠骨髓细胞模型显示出p18ink4c-/-造血干细胞自我更新能力增强。我们的实验以p18ink4c为目标,确定下调p18ink4c(?)的最佳siRNA序列,将该序列克隆到真核细胞siRNA表达载体,并构建包装pl8ink4c-siRNA腺病毒载体。实验分别采用合成siRNA、p18ink4c-siRNA质粒和p18ink4c-siRNA腺病毒载体转染人脐血纯化CD34+细胞。结果发现运用siRNA技术可以下调人脐血CD34+细胞的p18ink4c表达,扩增细胞数,降低CD34+的减少。但是转染效率较低,难以满足临床的需要。这两部分实验,分别通过改变扩增脐血造血干祖细胞的培养环境和下调脐血干祖细胞细胞周期负向调控分子,达到或部分达到了扩增脐血造血干祖细胞的目的。其中三维藻酸盐培养系统对脐血干祖细胞的扩增达到理想结果,国内外迄今无相同报道。而目前对造血干祖细胞转基因技术的转染效率不高,限制了下调p18ink4c分子扩增造血干祖细胞的效率,还有待于今后转染技术的完善和突破。
[Abstract]:Umbilical cord blood progenitor cells is one of the sources of clinical good stem. However, umbilical blood can provide the hematopoietic stem progenitor cell number, limiting the scope of application of cord blood transplantation. In order to meet the needs of adolescent and adult patients, must be in before transplantation in vitro amplification of cord blood hematopoietic stem progenitor cells. The experimental methods at present the in vitro expansion of hematopoietic stem and progenitor cells is a single progenitor cell suspension culture of hematopoietic stem, joint action depends on the higher dose of cytokines in the culture system. The amplified cells cannot be long-term hematopoietic reconstruction. Because of this, the development of new methods in vitro expansion of hematopoietic stem and progenitor cells is very necessary.
The first part, the alginate three-dimensional culture system in vitro amplification of human umbilical cord blood hematopoietic stem and progenitor cells in the in vivo survival of hematopoietic stem and progenitor cells in the bone marrow microenvironment, hematopoietic cells, between hematopoietic cells and stromal cells, interaction between cells and the extracellular matrix is one of the factors of stem cell self-renewal. Three dimensional culture system to maintain design and Simulation of bone marrow microenvironment. Our experimental design of alginate three-dimensional culture system, packaging of cord blood mononuclear cells were cultured in Petri dish static system and Synthecon RWV rotary culture system, and the conventional training system (2D system). The results showed that compared with the conventional culture system culture of umbilical cord blood mononuclear cells on cytokines in low concentration the total number of cells decreased, CD34+ cells decreased significantly. The three-dimensional static culture system in cytokine culture conditions under low concentration, the total number of cells increased, CD34+ cells Cell culture system increased significantly. The 3-D rotation can get similar results in lower concentrations of cytokines. The granulocyte macrophage colony formation in three-dimensional culture system of cells was also significantly increased in transplanted NOD/SCID mice transplanted cells count less, better in vivo engraftment and hematopoietic reconstruction.
The second part, down-regulation of cyclin dependent kinase inhibitor p18INK4C on human umbilical cord blood hematopoietic stem progenitor cells proliferation and differentiation of cells is tightly regulated cell cycle regulation of cell cycle. There are three kinds of important points, cyclins, cyclin dependent kinase and cyclin dependent kinase inhibitor CKI.p18ink4c belong to CKI, its role in the early G1 phase of the cell cycle, inhibiting the activity of the cyclinD-CDK4/6 complex, the cells were arrested in GO phase of.P18ink4c-/- mice showed widespread hyperplasia and organomegaly, p18ink4c-/- hematopoietic stem cell self-renewal ability enhancement of normal mice p18ink4c-/- transplantation of bone marrow cells in mice model. Our experiments using p18INK4C as the target, to determine the down-regulation of p18INK4C (?) the optimal siRNA sequence, the sequence was cloned into the siRNA eukaryotic expression vector, and construct pl8ink4c-siR packaging NA adenovirus vector. Experiments were used to synthesize siRNA, p18ink4c-siRNA plasmid and p18ink4c-siRNA adenovirus transfection of human umbilical cord blood purified CD34+ cells. The results showed that the expression of human umbilical cord blood CD34+ cells can be reduced with the use of siRNA technology p18INK4C, amplified cell number decreased CD34+ decreased. But the transfection efficiency is low and difficult to meet the clinical needs.
The two part of the experiment, the culture environment respectively by changing the expansion of hematopoietic stem and progenitor cells and down-regulation of cord blood stem / progenitor cell cycle negative regulatory molecules, achieved or partially achieved the expansion of hematopoietic stem and progenitor cells. The three-dimensional alginate culture system of umbilical cord blood stem cell amplification to achieve the desired results, both at home and abroad so far no the same report. At present on hematopoietic stem progenitor cell transfection efficiency of transgenic technology is not high, the efficiency is limited by the down-regulation of p18INK4C molecular amplification of hematopoietic stem and progenitor cells, and has yet to be perfect through transfection.

【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R329

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